Digestive malignant neoplasms including esophageal, gastric, and colorectal cancer are the most common cause of cancer induced death in the world. For example, colorectal cancer (CRC) is the third most common cancer in men (746,000 cases, 10.0% of total cancer cases) and the second most common cancer in women (614,000 cases, 9.2%) worldwide and it is the third most frequent cause of cancer mortality in the world for both genders. The European Union alone recorded 345,000 colorectal cancer incidences and 152,000 deaths from colorectal cancer in 2012.
To decrease mortality rates, various platforms for early stage cancer detection have been developed and tested to complete diagnostic evaluations on recruited eligible populations. However, it is difficult to cure gastrointestinal cancers detected with these platforms because they are often discovered at the progressive state. Moreover, the majority of the symptoms associated with gastrointestinal cancers do not manifest themselves until late in their development. Accordingly, the performance status of patients with gastrointestinal cancers and their overall prognosis needs to be improved.
For example, although screening programs for colorectal cancer have become more prevalent and survival rates have gone up within last 30 years, only around 40-44% of the cancers are detected in an early, localized stage, likely due to the lack of sensitivity of most of these screening programs. The general recommendation both in the U.S. and Europe is that individuals with average risk for colorectal cancer start regular screening either with colonoscopy or fecal blood test at the age of 50. However, the cancer incidence is increasing also among younger adults.
Understanding alterations in metabolic profiles in the colon that occur with tumor onset and progression could lead to better diagnostic tests as well as uncover new approaches for treatment or even prevention of CRC. Most CRCs are believed to originate from adenomatous polyps that acquire distinct mutations and accumulate other molecular alterations that allow them to progress through distinct histopathologic stages before becoming invasive carcinomas. Some adenomatous polyps do not progress to invasive tumors. Thus, the availability of a metabolic fingerprint that could distinguish a polyp that is likely to progress from one that will not progress could guide the frequency of screening colonoscopies and other preventative measures.
Metabolomics, which is understood as the quantitative measurement of all (or a certain percentage of all, e.g., most) low-molecular-weight metabolites in an organism at a specified time under specific environmental conditions, has been shown to be an effective tool for disease diagnosis, biomarker screening, and characterization of biological pathways.
Metabolites are the end products of cellular processes and their concentrations reflect the functional status of the organism and thus they are closely related to the observed phenotype. Perturbations in biological pathways can amplify the concentration changes of metabolites, making small molecule metabolites very attractive biomarkers of disease detection.
Altered metabolism is a cancer hallmark, resulting from changes in signaling pathways, protein expression, and other molecular mechanisms. Altered metabolism also reflects specific biochemical adaptations during carcinogenesis, which may confer malignant cells' survival advantages.
Two of the most prominent technologies for metabolite detection and quantification are nuclear magnetic resonance (NMR) and mass spectrometry (e.g., coupled to liquid chromatograph—LC-MS, gas chromatograph—GC-MS or direct analysis—DESI, DART). Both NMS and MS technologies are widely used in research and clinical settings.
Mass spectrometry is essentially a technique for “weighing” molecules. It is based upon the motion of charged particles, called ions, in an electric or magnetic field. The mass to charge ratio (m/z) of a particular ion affects this motion. Since the charge of an electron is known, then the mass to charge ratio is a measurement of an ion's mass. Mass spectrometry allows scanning for a wide range of metabolites. Data can include several thousands of detected metabolic events and thus creates a large pool for potential biomarker selection.
Several challenges exist in applying mass spectrometry measurements of metabolites and lipids in order to identify the presence or risk of colorectal cancer and/or adenomatous polyps in a subject.
First, the sensitivity of mass spectrometry instrumentation has only recently, over the past 5 years, become sufficient to enable global profiling of different biomaterials. Accordingly, metabolite based diagnostics that look into the whole human metabolon are still in their developmental infancy.
Secondly, accurate measurements of metabolites generally requires an internal standard for each marker of interest. Such standards for metabolites may not commercially available, and accordingly must be synthesized. Accordingly, performing accurate measurements of metabolites requires significantly more than just measuring a given metabolite. The additional step and capability of synthesizing an internal standard is required.
Another challenge to the use of metabolite and lipid based biomarker detection is the sensitivity of metabolomics to population differences (e.g. population bias). Although metabolite profiles/fingerprints have the potential to act as indicators of disease, they are also very sensitive to minor differences in the biological background, and therefore also vary significantly across different populations. Therefore, a challenge in metabolomics based diagnostics is finding a method that is sensitive to the disease of interest, while at the same time robust to variations across e.g. different populations.
Likewise, metabolites and lipids are quite sensitive molecules to the pre-analytical treatment of a sample, including sample collection method, storage conditions, and other preparation steps. As a result, measurements of metabolites and lipids are influenced by, e.g. hemolysis, lipemia, sample time at room temperature before serum extraction and freezing, and the freeze-thaw cycles applied to the samples. Accordingly, sample collection and preparation methods that comprise appropriate quality controls and/or yield consistent measurements of target biomarkers must be developed and employed.
There is a need for diagnostics to identify the presence, stage, and/or risk of colorectal cancer and/or adenomatous polyps in a subject. Metabolomics and mass spectrometry based diagnostics is a promising approach, but requires identifying an appropriate panel of markers that are sensitive to the presence of colorectal cancer and/or adenomatous polyps in a subject, while at the same time robust to population variations, such as e.g. gender, age, ethnicity. Additionally, appropriate internal standards, and sample handling and quality control methods that enable accurate and reliable measurements are required.